Anti-obesity proteins

ABSTRACT

The present invention provides anti-obesity proteins, which when administered to a patient regulate fat tissue. Accordingly, such agents allow patients to overcome their obesity handicap and live normal lives with much reduced risk for type II diabetes, cardiovascular disease and cancer.

This application is a continuation-in-part of DiMarchi et al., U.S. Ser.No. 08/381,661, filed Jan. 31, 1995, docket number X-9947 now abandoned.

FIELD OF THE INVENTION

The present invention is in the field of human medicine, particularly inthe treatment of obesity and disorders associated with obesity. Mostspecifically the invention relates to anti-obesity proteins that whenadministered to a patient regulate fat tissue.

BACKGROUND OF THE INVENTION

Obesity, and especially upper body obesity, is a common and very seriouspublic health problem in the United States and throughout the world.According to recent statistics, more than 25% of the United Statespopulation and 27% of the Canadian population are over weight.Kuczmarski, Amer. J. of Clin. Nut. 52: 495S-502S (1992); Reeder et. al.,Can. Med. Ass. J., 23:226-233 (1992). Upper body obesity is thestrongest risk factor known for type II diabetes mellitus, and is astrong risk factor for cardiovascular disease and cancer as well. Recentestimates for the medical cost of obesity are $150,000,000,000 worldwide. The problem has become serious enough that the surgeon general hasbegun an initiative to combat the ever increasing adiposity rampant inAmerican society.

Much of this obesity induced pathology can be attributed to the strongassociation with dyslipidemia, hypertension, and insulin resistance.Many studies have demonstrated that reduction in obesity by diet andexercise reduces these risk factors dramatically. Unfortunately thesetreatments are largely unsuccessful with a failure rate reaching 95%.This failure may be due to the fact that the condition is stronglyassociated with genetically inherited factors that contribute toincreased appetite, preference for highly caloric foods, reducedphysical activity, and increased lipogenic metabolism. This indicatesthat people inheriting these genetic traits are prone to becoming obeseregardless of their efforts to combat the condition. Therefore, a newpharmacological agent that can correct this adiposity handicap and allowthe physician to successfully treat obese patients in spite of theirgenetic inheritance is needed.

The ob/ob mouse is a model of obesity and diabetes that is known tocarry an autosomal recessive trait linked to a mutation in the sixthchromosome. Recently, Yiying Zhang and co-workers published thepositional cloning of the mouse gene linked with this condition. YiyingZhang et al. Nature 372:425-32 (1994). This report disclosed a genecoding for a 167 amino acid protein with a 21 amino acid signal peptidethat is exclusively expressed in adipose tissue. The report continues todisclose that a mutation resulting in the conversion of a codon forarginine at position 105 to a stop codon results in the expression of atruncated protein, which presumably is inactive.

Physiologist have postulated for years that, when a mammal overeats, theresulting excess fat signals to the brain that the body is obese which,in turn, causes the body to eat less and burn more fuel. G. R. Hervey,Nature 227:629-631 (1969). This "feedback" model is supported byparabiotic experiments, which implicate a circulating hormonecontrolling adiposity. Based on this model, the protein, which isapparently encoded by the ob gene, is now speculated to be an adiposityregulating hormone.

Pharmacological agents which are biologically active and mimic theactivity of this protein are useful to help patients regulate theirappetite and metabolism and thereby control their adiposity. Until thepresent invention, such a pharmacological agent was unknown.

The present invention provides biologically active anti-obesityproteins. Such agents therefore allow patients to overcome their obesityhandicap and live normal lives with a more normalized risk for type IIdiabetes, cardiovascular disease and cancer.

SUMMARY OF INVENTION

The present invention is directed to a biologically active anti-obesityprotein of the Formula (I): ##STR1## wherein: Xaa at position 4 of SEQID NO: 1 is Gln or Glu;

Xaa at position 7 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 22 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 27 of SEQ ID NO: 1 is Thr or Ala;

Xaa at position 28 of SEQ ID NO: 1 is Gin, Glu or absent;

Xaa at position 34 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 54 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 56 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 62 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 63 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 68 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 72 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 75 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 78 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 82 of SEQ ID NO: 1 is Ash, Asp or Gln;

Xaa at position 100 of SEQ ID NO: 1 is Gln, Trp, Tyr, Phe, Ile, Val, orLeu;

Xaa at position 130 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 134 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 136 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 138 of SEQ ID NO: 1 is Trp or Gln; and

Xaa at position 1 of SEQ ID NO: 2 is Gln or Glu.

The invention further provides a method of treating obesity, whichcomprises administering to a mammal in need thereof a protein of theFormula (I).

The invention further provides a pharmaceutical formulation, whichcomprises a protein of the Formula (I) together with one or morepharmaceutical acceptable diluents, carriers or excipients therefor.

DETAILED DESCRIPTION

As noted above the present invention provides a protein of the Formula(I): ##STR2## wherein: Xaa at position 4 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 7 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 22 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 27 of SEQ ID NO: 1 is Thr or Ala;

Xaa at position 28 of SEQ ID NO: 1 is Gln, Glu or absent;

Xaa at position 34 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 54 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 56 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 62 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 63 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 68 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 72 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 75 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 78 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 82 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 100 of SEQ ID NO: 1 is Gln, Trp, Tyr, Phe, Ile, Val, orLeu;

Xaa at position 130 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 134 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 136 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 138 of SEQ ID NO: 1 is Trp or Gln; and

Xaa at position 1 of SEQ ID NO: 2 is Gln or Glu.

The preferred proteins of the present invention are those of Formula (I)wherein:

wherein:

Xaa at position 4 of SEQ ID NO: 1 is Gln;

Xaa at position 7 of SEQ ID NO: 1 is Gln;

Xaa at position 22 of SEQ ID NO: 1 is Asn;

Xaa at position 27 of SEQ ID NO: 1 is Thr;

Xaa at position 28 of SEQ ID NO: 1 is Gln;

Xaa at position 34 of SEQ ID NO: 1 is Gln;

Xaa at position 54 of SEQ ID NO: 1 is Met;

Xaa at position 56 of SEQ ID NO: 1 is Gln;

Xaa at position 62 of SEQ ID NO: 1 is Gln;

Xaa at position 63 of SEQ ID NO: 1 is Gln;

Xaa at position 68 of SEQ ID NO: 1 is Met;

Xaa at position 72 of SEQ ID NO: 1 is Asn;

Xaa at position 75 of SEQ ID NO: 1 is Gln;

Xaa at position 78 of SEQ ID NO: 1 is Asn;

Xaa at position 82 of SEQ ID NO: 1 is Asn;

Xaa at position 100 of SEQ ID NO: 1 is Trp;

Xaa at position 130 of SEQ ID NO: 1 is Gln;

Xaa at position 134 of SEQ ID NO: 1 is Gln;

Xaa at position 136 of SEQ ID NO: 1 is Met;

Xaa at position 138 of SEQ ID NO: 1 is Trp; and

Xaa at position 1 of SEQ ID NO: 2 is Gln.

The amino acids abbreviations are accepted by the United States Patentand Trademark Office as set forth in 37 C.F.R. § 1.822 (b)(2) (1993).One skilled in the art would recognize that certain amino acids areprone to rearrangement. For example, Asp may rearrange to aspartimideand isoasparigine as described in I. Schon et al., Int. J. PeptideProtein Res. 14:485-94 (1979) and references cited therein. Theserearrangement derivatives are included within the scope of the presentinvention. Unless otherwise indicated the amino acids are in the Lconfiguration.

For purposes of the present invention, as disclosed and claimed herein,the following terms and abbreviations are defined as follows:

SEQ ID NO:1 properly crosslinked to SEQ ID NO: 2 refers to the formationof a disulfide bond between cysteine residues at position 96 of SEQ IDNO: 1 and position 8 of SEQ ID NO: 2 as depicted in Formula I.

Base pair (bp)--refers to DNA or RNA. The abbreviations A,C,G, and Tcorrespond to the 5'-monophosphate forms of the nucleotides(deoxy)adenine, (deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine,respectively, when they occur in DNA molecules. The abbreviations U,C,G,and T correspond to the 5'-monophosphate forms of the nucleosidesuracil, cytidine, guanine, and thymine, respectively when they occur inRNA molecules. In double stranded DNA, base pair may refer to apartnership of A with T or C with G. In a DNA/RNA heteroduplex, basepair may refer to a partnership of T with U or C with G.

Chelating Peptide--An amino acid sequence capable of complexing with amultivalent metal ion.

DNA--Deoyxribonucleic acid.

EDTA--an abbreviation for ethylenediamine tetraacetic acid.

ED₅₀ --an abbreviation for half-maximal value.

FAB-MS--an abbreviation for fast atom bombardment mass spectrometry.

Immunoreactive Protein(s)--a term used to collectively describeantibodies, fragments of antibodies capable of binding antigens of asimilar nature as the parent antibody molecule from which they arederived, and single chain polypeptide binding molecules as described inPCT Application No. PCT/US 87/02208, International Publication No. WO88/01649.

mRNA--messenger RNA.

MWCO--an abbreviation for molecular weight cut-off.

Plasmid--an extrachromosomal self-replicating genetic element.

PMSF--an abbreviation for phenylmethylsulfonyl fluoride.

Reading frame--the nucleotide sequence from which translation occurs"read" in triplets by the translational apparatus of tRNA, ribosomes andassociated factors, each triplet corresponding to a particular aminoacid. Because each triplet is distinct and of the same length, thecoding sequence must be a multiple of three. A base pair insertion ordeletion (termed a frameshift mutation) may result in two differentproteins being coded for by the same DNA segment. To insure againstthis, the triplet codons corresponding to the desired polypeptide mustbe aligned in multiples of three from the initiation codon, i.e. thecorrect "reading frame" must be maintained. In the creation of fusionproteins containing a chelating peptide, the reading frame of the DNAsequence encoding the structural protein must be maintained in the DNAsequence encoding the chelating peptide.

Recombinant DNA Cloning Vector--any autonomously replicating agentincluding, but not limited to, plasmids and phages, comprising a DNAmolecule to which one or more additional DNA segments can or have beenadded.

Recombinant DNA Expression Vector--any recombinant DNA cloning vector inwhich a promoter has been incorporated.

Replicon--A DNA sequence that controls and allows for autonomousreplication of a plasmid or other vector.

RNA--ribonucleic acid.

RP-HPLC--an abbreviation for reversed-phase high performance liquidchromatography.

Transcription--the process whereby information contained in a nucleotidesequence of DNA is transferred to a complementary RNA sequence.

Translation--the process whereby the genetic information of messengerRNA is used to specify and direct the synthesis of a polypeptide chain.

Tris--an abbreviation for tris(hydroxymethyl)aminomethane.

Treating--describes the management and care of a patient for the purposeof combating the disease, condition, or disorder and includes theadministration of a compound of present invention to prevent the onsetof the symptoms or complications, alleviating the symptoms orcomplications, or eliminating the disease, condition, or disorder.Treating obesity therefor includes the inhibition of food intake, theinhibition of weight gain, and inducing weight loss in patients in needthereof.

Vector--a replicon used for the transformation of cells in genemanipulation bearing polynucleotide sequences corresponding toappropriate protein molecules which, when combined with appropriatecontrol sequences, confer specific properties on the host cell to betransformed. Plasmids, viruses, and bacteriophage are suitable vectors,since they are replicons in their own right. Artificial vectors areconstructed by cutting and joining DNA molecules from different sourcesusing restriction enzymes and ligases. Vectors include Recombinant DNAcloning vectors and Recombinant DNA expression vectors.

X-gal--an abbreviation for 5-bromo-4-chloro-3-idolyl beta-D-galactoside.

SEQ ID NO: 1 refers to the sequence set forth in the sequence listingand means an anti-obesity protein of the formula: ##STR3## wherein: Xaaat position 4 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 7 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 22 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 27 of SEQ ID NO: 1 is Thr or Ala;

Xaa at position 28 of SEQ ID NO: 1 is Gln, Glu or absent;

Xaa at position 34 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 54 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 56 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 62 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 63 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 68 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly;

Xaa at position 72 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 75 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 78 of SEQ ID NO: 1 is Ash, Asp or Gln;

Xaa at position 82 of SEQ ID NO: 1 is Asn, Asp or Gln;

Xaa at position 100 of SEQ ID NO: 1 is Gln, Trp, Tyr, Phe, Ile, Val, orLeu;

Xaa at position 130 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 134 of SEQ ID NO: 1 is Gln or Glu;

Xaa at position 136 of SEQ ID NO: 1 is Met, methionine sulfoxide, Leu,Ile, Val, Ala, or Gly; and

Xaa at position 138 of SEQ ID NO: 1 is Trp or Gln.

SEQ ID NO: 2 refers to the sequence set forth in the sequence listingand means an anti-obesity protein of the formula: ##STR4## wherein: Xaaat position 1 of SEQ ID NO: 2 is Gln or Glu.

Yiying Zhang et al. in Nature 372:425-32 (December 1994) report thecloning of the murine obese (ob) mouse gene and present mouse DNA andthe naturally occurring amino acid sequence of the obesity protein forthe mouse and human. This protein is speculated to be a hormone that issecreted by fat cells and controls body weight.

The present invention provides biologically active proteins that provideeffective treatment for obesity. Many of the claimed proteins offeradditional advantages of better absorption characteristics allowingadministration nasally, bronchally, transdermally, or parentally. Inaddition, the claimed proteins improve in vivo stability or increasedbiological half-life.

The claimed proteins ordinarily are prepared by modification of the DNAencoding the claimed protein and thereafter expressing the DNA inrecombinant cell culture. Techniques for making substitutional mutationsat predetermined sites in DNA having a known sequence are well known,for example M13 primer mutagenesis. The mutations that might be made inthe DNA encoding the present anti-obesity proteins must not place thesequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. SeeDeBoer et al., EP 75,444A (1983).

The compounds of the present invention may be produced either byrecombinant DNA technology or well known chemical procedures, such assolution or solid-phase peptide synthesis, or semi-synthesis in solutionbeginning with protein fragments coupled through conventional solutionmethods.

A. Solid Phase

The synthesis of the claimed protein may proceed by solid phase peptidesynthesis or by recombinant methods. The principles of solid phasechemical synthesis of polypeptides are well known in the art and may befound in general texts in the area such as Dugas, H. and Penney, C.,Bioorganic Chemistry Springer-Verlag, New York, pgs. 54-92 (1981). Forexample, peptides may be synthesized by solid-phase methodologyutilizing an PE-Applied Biosystems 430A peptide synthesizer(commercially available from Applied Biosystems, Foster City Calif.) andsynthesis cycles supplied by Applied Biosystems. Boc amino acids andother reagents are commercially available from PE-Applied Biosystems andother chemical supply houses. Sequential Boc chemistry using doublecouple protocols are applied to the starting p-methyl benzhydryl amineresins for the production of C-terminal carboxamides. For the productionof C-terminal acids, the corresponding PAM resin is used. Arginine,Asparagine, Glutamine, Histidine and Methionine are coupled usingpreformed hydroxy benzotriazole esters. The following side chainprotection may be used:

Arg, Tosyl

Asp, cyclohexyl or benzyl

Cys, 4-methylbenzyl

Glu, cyclohexyl

His, benzyloxymethyl

Lys, 2-chlorobenzyloxycarbonyl

Met, sulfoxide

Ser, Benzyl

Thr, Benzyl

Trp, formyl

Tyr, 4-bromo carbobenzoxy

Boc deprotection may be accomplished with trifluoroacetic acid (TFA) inmethylene chloride. Formyl removal from Trp is accomplished by treatmentof the peptidyl resin with 20% piperidine in dimethylformamide for 60minutes at 4° C. Met(O) can be reduced by treatment of the peptidylresin with TFA/dimethylsulfide/conHCl (95/5/1) at 25° C. for 60 minutes.Following the above pre-treatments, the peptides may be furtherdeprotected and cleaved from the resin with anhydrous hydrogen fluoridecontaining a mixture of 10% m-cresol or m-cresol/10% p-thiocresol orm-cresol/p-thiocresol/dimethylsulfide. Cleavage of the side chainprotecting group(s) and of the peptide from the resin is carried out atzero degrees Centigrade or below, preferably -20° C. for thirty minutesfollowed by thirty minutes at 0° C. After removal of the HF, thepeptide/resin is washed with ether. The peptide is extracted withglacial acetic acid and lyophilized. Purification is accomplished byreverse-phase C18 chromatography (Vydac) column in 0.1% TFA with agradient of increasing acetonitrile concentration.

One skilled in the art recognizes that the solid phase synthesis couldalso be accomplished using the FMOC strategy and a TFA/scavengercleavage mixture.

B. Recombinant Synthesis

The claimed proteins may also be produced by recombinant methods.Recombinant methods are preferred if a high yield is desired. The basicsteps in the recombinant production of protein include:

a) construction of a synthetic or semi-synthetic (or isolation fromnatural sources) DNA encoding the claimed protein,

b) integrating the coding sequence into an expression vector in a mannersuitable for the expression of the protein either alone or as a fusionprotein,

c) transforming an appropriate eukaryotic or prokaryotic host cell withthe expression vector, and

d) recovering and purifying the recombinantly produced protein.

2.a. Gene Construction

Synthetic genes, the in vitro or in vivo transcription and translationof which will result in the production of the protein may be constructedby techniques well known in the art. Owing to the natural degeneracy ofthe genetic code, the skilled artisan will recognize that a sizable yetdefinite number of DNA sequences may be constructed which encode theclaimed proteins. In the preferred practice of the invention, synthesisis achieved by recombinant DNA technology.

Methodology of synthetic gene construction is well known in the art. Forexample, see Brown, et al. (1979) Methods in Enzymology, Academic Press,N.Y., Vol. 68, pgs. 109-151. The DNA sequence corresponding to thesynthetic claimed protein gene may be generated using conventional DNAsynthesizing apparatus such as the Applied Biosystems Model 380A or 380BDNA synthesizers (commercially available from Applied Biosystems, Inc.,850 Lincoln Center Drive, Foster City, Calif. 94404).

It may desirable in some applications to modify the coding sequence ofthe claimed protein so as to incorporate a convenient protease sensitivecleavage site, e.g., between the signal peptide and the structuralprotein facilitating the controlled excision of the signal peptide fromthe fusion protein construct.

The gene encoding the claimed protein may also be created by usingpolymerase chain reaction (PCR). The template can be a cDNA library(commercially available from CLONETECH or STRATAGENE) or mRNA isolatedfrom human adipose tissue. Such methodologies are well known in the artManiatis, et al. Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989).

2.b. Direct Expression or Fusion Protein

The claimed protein may be made either by direct expression or as fusionprotein comprising the claimed protein followed by enzymatic or chemicalcleavage. A variety of peptidases (e.g. trypsin) which cleave apolypeptide at specific sites or digest the peptides from the amino orcarboxy termini (e.g. diaminopeptidase) of the peptide chain are known.Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave apolypeptide chain at specific sites. The skilled artisan will appreciatethe modifications necessary to the amino acid sequence (and synthetic orsemi-synthetic coding sequence if recombinant means are employed) toincorporate site-specific internal cleavage sites. See e.g., Carter P.,Site Specific Proteolysis of Fusion Proteins, Ch. 13 in ProteinPurification: From Molecular Mechanisms to Large Scale Processes,American Chemical Soc., Washington, D.C. (1990).

2.c. Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and religated in the formdesired to form the plasmids required.

To effect the translation of the desired protein, one inserts theengineered synthetic DNA sequence in any of a plethora of appropriaterecombinant DNA expression vectors through the use of appropriaterestriction endonucleases. The claimed protein is a relatively largeprotein. A synthetic coding sequence is designed to possess restrictionendonuclease cleavage sites at either end of the transcript tofacilitate isolation from and integration into these expression andamplification and expression plasmids. The isolated cDNA coding sequencemay be readily modified by the use of synthetic linkers to facilitatethe incorporation of this sequence into the desired cloning vectors bytechniques well known in the art. The particular endonucleases employedwill be dictated by the restriction endonuclease cleavage pattern of theparent expression vector to be employed. The choice of restriction sitesare chosen so as to properly orient the coding sequence with controlsequences to achieve proper in-frame reading and expression of theclaimed protein.

In general, plasmid vectors containing promoters and control sequenceswhich are derived from species compatible with the host cell are usedwith these hosts. The vector ordinarily carries a replication site aswell as marker sequences which are capable of providing phenotypicselection in transformed cells. For example, E. coli is typicallytransformed using DBR322, a plasmid derived from an E. coli species(Bolivar, et al., Gene 2:95 (1977)). Plasmid pBR322 contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells. The pBR322 plasmid, or other microbialplasmid must also contain or be modified to contain promoters and othercontrol elements commonly used in recombinant DNA technology.

The desired coding sequence is inserted into an expression vector in theproper orientation to be transcribed from a promoter and ribosomebinding site, both of which should be functional in the host cell inwhich the protein is to be expressed. An example of such an expressionvector is a plasmid described in Belagaje et al., U.S. Pat. No.5,304,493, the teachings of which are herein incorporated by reference.The gene encoding A-C-B proinsulin described in U.S. Pat. No. 5,304,493can be removed from the plasmid pRB182 with restriction enzymes NdeI andBamHI. The genes encoding the protein of the present invention can beinserted into the plasmid backbone on a NdeI/BamHI restriction fragmentcassette.

2.d. Procaryotic Expression

In general, procaryotes are used for cloning of DNA sequences inconstructing the vectors useful in the invention. For example, E. coliK12 strain 294 (ATCC No. 31446) is particularly useful. Other microbialstrains which may be used include E. coli B and E. coli X1776 (ATCC No.31537). These examples are illustrative rather than limiting.

Prokaryotes also are used for expression. The aforementioned strains, aswell as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such asBacillus subtilis, and other enterobacteriaceae such as Salmonellatyphimurium or Serratia marcescans, and various pseudomonas species maybe used. Promoters suitable for use with prokaryotic hosts include theβ-lactamase (vector pGX2907 [ATCC 39344] contains the replicon andβ-lactamase gene) and lactose promoter systems (Chang et al., Nature,275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), alkalinephosphatase, the tryptophan (trp) promoter system (vector pATHi [ATCC37695] is designed to facilitate expression of an open reading frame asa trpE fusion protein under control of the trp promoter) and hybridpromoters such as the tac promoter (isolatable from plasmid pDR540ATCC-37282). However, other functional bacterial promoters, whosenucleotide sequences are generally known, enable one of skill in the artto ligate them to DNA encoding the protein using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno sequence operably linked tothe DNA encoding protein.

2.e. Eucaryotic Expression

The protein may be recombinantly produced in eukaryotic expressionsystems. Preferred promoters controlling transcription in mammalian hostcells may be obtained from various sources, for example, the genomes ofviruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. β-actin promoter. The earlyand late promoters of the SV40 virus are conveniently obtained as anSV40 restriction fragment which also contains the SV40 viral origin ofreplication. Fiers, et al., Nature, 273:113 (1978). The entire SV40genome may be obtained from plasmid pBRSV, ATCC 45019. The immediateearly promoter of the human cytomegalovirus may be obtained from plasmidpCMBD (ATCC 77177). Of course, promoters from the host cell or relatedspecies also are useful herein.

Transcription of a DNA encoding the claimed protein by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about 10-300 bp, thatact on a promoter to increase its transcription. Enhancers arerelatively orientation and position independent having been found 5'(Laimins, L. et al., PNAS 78:993 (1981)) and 3' (Lusky, M. L., et al.,Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within anintron (Banerji, J. L. et al., Cell 33:729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4:1293(1984)). Many enhancer sequences are now known from mammalian genes(globin, RSV, SV40, EMC, elastase, albumin, afetoprotein and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 late enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription which may affect mRNA expression. These regions aretranscribed as polyadenylated segments in the untranslated portion ofthe mRNA encoding protein. The 3' untranslated regions also includetranscription termination sites.

Expression vectors may contain a selection gene, also termed aselectable marker. Examples of suitable selectable markers for mammaliancells are dihydrofolate reductase (DHFR, which may be derived from theBglII/HindIII restriction fragment of pJOD-10 [ATCC 68815]), thymidinekinase (herpes simplex virus thymidine kinase is contained on the BamHIfragment of vP-5 clone [ATCC 2028]) or neomycin (G418) resistance genes(obtainable from pNN414 yeast artificial chromosome vector [ATCC37682]). When such selectable markers are successfully transferred intoa mammalian host cell, the transfected mammalian host cell can surviveif placed under selective pressure. There are two widely used distinctcategories of selective regimes. The first category is based on a cell'smetabolism and the use of a mutant cell line which lacks the ability togrow without a supplemented media. Two examples are: CHO DHFR⁻ cells(ATCC CRL-9096) and mouse LTK⁻ cells (L-M(TK-) ATCC CCL-2.3). Thesecells lack the ability to grow without the addition of such nutrients asthymidine or hypoxanthine. Because these cells lack certain genesnecessary for a complete nucleotide synthesis pathway, they cannotsurvive unless the missing nucleotides are provided in a supplementedmedia. An alternative to supplementing the media is to introduce anintact DHFR or TK gene into cells lacking the respective genes, thusaltering their growth requirements. Individual cells which were nottransformed with the DHFR or TK gene will not be capable of survival innonsupplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, Southern P. and Berg,P., J. Molec. Appl. Genet. 1:327 (1982), mycophenolic acid, Mulligan, R.C. and Berg, P. Science 209:1422 (1980), or hygromycin, Sugden, B. etal., Mol Cell. Biol. 5:410-413 (1985). The three examples given aboveemploy bacterial genes under eukaryotic control to convey resistance tothe appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolicacid) or hygromycin, respectively.

A preferred vector for eucaryotic expression is pRc/CMV. pRc/CMV iscommercially available from Invitrogen Corporation, 3985 Sorrento ValleyBlvd., San Diego, Calif. 92121. To confirm correct sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K12strain DH5a (ATCC 31446) and successful transformants selected byantibiotic resistance where appropriate. Plasmids from the transformantsare prepared, analyzed by restriction and/or sequence by the method ofMessing, et al., Nucleic Acids Res. 4:309 (1981).

Host cells may be transformed with the expression vectors of thisinvention and cultured in conventional nutrient media modified as isappropriate for inducing promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan. Thetechniques of transforming cells with the aforementioned vectors arewell known in the art and may be found in such general references asManiatis, et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989), or Current Protocols in Molecular Biology (1989) andsupplements.

Preferred suitable host cells for expressing the vectors encoding theclaimed proteins in higher eukaryotes include: African green monkeykidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651);transformed human primary embryonal kidney cell line 293, (Graham, F. L.et al., J. Gen Virol. 36:59-72 (1977), Virology 77:319-329, Virology86:10-21); baby hamster kidney cells (BHK-21(C-13), ATCC CCL-10,Virology 16:147 (1962)); chinese hamster ovary cells CHO-DHFR⁻ (ATCCCRL-9096), mouse Sertoli cells (TM4, ATCC CRL-1715, Biol. Reprod.23:243-250 (1980)); african green monkey kidney cells (VERO 76, ATCCCRL-1587); human cervical epitheloid carcinoma cells (HeLa, ATCC CCL-2);canine kidney cells (MDCK, ATCC CCL-34); buffalo rat liver cells (BRL3A, ATCC CRL-1442); human diploid lung cells (WI-38, ATCC CCL-75); humanhepatocellular carcinoma cells (Hep G2, ATCC HB-8065);and mouse mammarytumor cells (MMT 060562, ATCC CCL51).

2.f. Yeast Expression

In addition to prokaryotes, eukaryotic microbes such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used eukaryotic microorganism, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, (ATCC-40053, Stinchcomb, et al., Nature282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al.,Gene 10:157 (1980)) is commonly used. This plasmid already contains thetrp gene which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example ATCC no. 44076 orPEP4-1 (Jones, Genetics 85:12 (1977)).

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD ATCC53231 and described in U.S. Pat. No. 4,935,350, Jun. 19, 1990) or otherglycolytic enzymes such as enolase (found on plasmid pAC1 ATCC 39532),glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPC1ATCC 7090, 57091), zymomonas mobilis (U.S. Pat. No. 5,000,000 issuedMar. 19, 1991), hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPVATCC 39475, U.S. Pat. No. 4,840,896), glyceraldehyde 3-phosphatedehydrogenase, and enzymes responsible for maltose and galactose (GAL1found on plasmid pRY121 ATCC 37658) utilization. Suitable vectors andpromoters for use in yeast expression are further described in R.Hitzeman et al., European Patent Publication No. 73,657A. Yeastenhancers such as the UAS Gal from Saccharomyces cerevisiae (found inconjunction with the CYC1 promoter on plasmid YEpsec--hI1beta ATCC67024), also are advantageously used with yeast promoters.

The following examples are presented to further illustrate thepreparation of the claimed proteins. The scope of the present inventionis not to be construed as merely consisting of the following examples.

EXAMPLE 1

A DNA sequence encoding the following protein sequence: ##STR5## isobtained using standard PCR methodology. A forward primer (5'-GG GG CATATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC) SEQ ID NO:3 and areverse primer (5'-GG GG GGATC CTA TTA GCA CCC GGG AGA CAG GTC CAG CTGCCA CAA CAT) SEQ ID NO:4 is used to amplify sequences from a human fatcell library (commercially available from CLONETECH). The PCR product iscloned into PCR-Script (available from STRATAGENE) and sequenced.

EXAMPLE 2 Vector Construction

A plasmid containing the DNA sequence encoding the desired claimedprotein is constructed to include NdeI and BamHI restriction sites. Theplasmid carrying the cloned PCR product is digested with NdeI and BamHIrestriction enzymes. The small ˜450 bp fragment is gel-purified andligated into the vector pRB182 from which the coding sequence for A-C-Bproinsulin is deleted. The ligation products are transformed into E.coli DH10B (commercially available from GIBCO-BRL) and colonies growingon tryptone-yeast (DIFCO) plates supplemented with 10 μg/mL oftetracycline are analyzed. Plasmid DNA is isolated, digested with NdeIand BamHI and the resulting fragments are separated by agarose gelelectrophoresis. Plasmids containing the expected ˜450 bp NdeI to BaSHIfragment are kept. E. coli B BL21 (DE3) (commercially available fromNOVOGEN) are transformed with this second plasmid expression suitablefor culture for protein production.

The techniques of transforming cells with the aforementioned vectors arewell known in the art and may be found in such general references asManiatis, et al. (1988) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. or Current Protocols in Molecular Biology (1989) and supplements.The techniques involved in the transformation of E. coli cells used inthe preferred practice of the invention as exemplified herein are wellknown in the art. The precise conditions under which the transformed E.coli cells are cultured is dependent on the nature of the E. coli hostcell line and the expression or cloning vectors employed. For example,vectors which incorporate thermoinducible promoter-operator regions,such as the c1857 thermoinducible lambda-phage promoter-operator region,require a temperature shift from about 30 to about 40 degrees C. in theculture conditions so as to induce protein synthesis.

In the preferred embodiment of the invention E. coli K12 RV308 cells areemployed as host cells but numerous other cell lines are available suchas, but not limited to, E. coli K12 L201, L687, L693, L507, L640, L641,L695, L814 (E. coli B). The transformed host cells are then plated onappropriate media under the selective pressure of the antibioticcorresponding to the resistance gene present on the expression plasmid.The cultures are then incubated for a time and temperature appropriateto the host cell line employed.

Proteins which are expressed in high-level bacterial expression systemscharacteristically aggregate in granules or inclusion bodies whichcontain high levels of the overexpressed protein. Kreuger et al., inProtein Folding, Gierasch and King, eds., pgs 136-142 (1990), AmericanAssociation for the Advancement of Science Publication No. 89-18S,Washington, D.C. Such protein aggregates must be solubilized to providefurther purification and isolation of the desired protein product. Id. Avariety of techniques using strongly denaturing solutions such asguanidinium-HCl and/or weakly denaturing solutions such asdithiothreitol (DTT) are used to solubilize the proteins.

Gradual removal of the denaturing agents (often by dialysis) in asolution allows the denatured protein to assume its native conformation.The particular conditions for denaturation and folding are determined bythe particular protein expression system and/or the protein in question.

Preferably, the present proteins are expressed as Met-Arg-SEQ ID NO: 1so that the expressed proteins may be readily converted to the claimedprotein with Cathepsin C. The purification of proteins is by techniquesknown in the art and includes reverse phase chromatography, affinitychromatography, and size exclusion.

The claimed proteins may exist, particularly when formulated, as dimers,trimers, tetramers, and other multimers. Such multimers are includedwithin the scope of the present invention.

The present invention provides a method for treating obesity. The methodcomprises administering to the organism an effective amount ofanti-obesity protein in a dose between about 1 and 1000 μg/kg. Apreferred dose is from about 10 to 100 μg/kg of active compound. Atypical daily dose for an adult human is from about 0.5 to 100 mg. Inpracticing this method, compounds of the Formula (I) can be administeredin a single daily dose or in multiple doses per day. The treatmentregime may require administration over extended periods of time. Theamount per administered dose or the total amount administered will bedetermined by the physician and depend on such factors as the nature andseverity of the disease, the age and general health of the patient andthe tolerance of the patient to the compound.

The instant invention further provides pharmaceutical formulationscomprising compounds of the Formula (I). The proteins, preferably in theform of a pharmaceutically acceptable salt, can be formulated for nasal,bronchal, transdermal, or parenteral administration for the therapeuticor prophylactic treatment of obesity. For example, compounds of theFormula (I) can be admixed with conventional pharmaceutical carriers andexcipients. The compositions comprising claimed proteins contain fromabout 0.1 to 90% by weight of the active protein, preferably in asoluble form, and more generally from about 10 to 30%.

For intravenous (IV) use, the protein is administered in commonly usedintravenous fluid(s) and administered by infusion. Such fluids, forexample, physiological saline, Ringer's solution or 5% dextrose solutioncan be used.

For intramuscular preparations, a sterile formulation, preferably asuitable soluble salt form of a protein of the Formula (I), for examplethe hydrochloride salt, can be dissolved and administered in apharmaceutical diluent such as pyrogen-free water (distilled),physiological saline or 5% glucose solution. A suitable insoluble formof the compound may be prepared and administered as a suspension in anaqueous base or a pharmaceutically acceptable oil base, e.g. an ester ofa long chain fatty acid such as ethyl oleate.

It may also be desirable to administer the compounds of Formula (I)intranasally. Formulations useful in the intranasal absorption ofproteins are well known in the art. Nasal formulations comprise theprotein and carboxyvinyl polymer preferably selected from the groupcomprising the acrylic acid series hydrophilic crosslinked polymer, e.g.carbopole 934; 940, 941 (Goodrich Co.). The polymer acceleratesabsorption of the protein, and gives suitable viscosity to preventdischarge from nose. Suitable content of the polymer is 0.05-2 weight %.By neutralisation of the polymer with basic substance, thickening effectis increased. The amount of active compound is commonly 0.1-10%. Thenasal preparation may be in drop form, spraying applicator or aerosolform.

The ability of the present compounds to treat obesity is demonstrated invivo as follows:

Biological Testing for Anti-Obesity Proteins

Parabiotic experiments suggest that a protein is released by peripheraladipose tissue and that the protein is able to control body weight gainin normal, as well as obese mice. Therefore, the most closely relatedbiological test is to inject the test article by any of several routesof administration (e.g. i.v., s.c., i.p., or by minipump or cannula) andthen to monitor food and water consumption, body weight gain, plasmachemistry or hormones (glucose, insulin, ACTH, corticosterone, GH, T4)over various time periods.

Suitable test animals include normal mice (ICR, etc.) and obese mice(ob/ob, Avy/a, KK-Ay, tubby, fat). The mouse model of obesity anddiabetes is generally accepted in the art as being indicative of theobesity condition. Controls for non-specific effects for theseinjections are done using vehicle with or without the active agent ofsimilar composition in the same animal monitoring the same parameters orthe active agent itself in animals that are thought to lack the receptor(db/db mice, fa/fa or cp/cp rats). Proteins demonstrating activity inthese models will demonstrate similar activity in other mammals,particularly humans.

Since the target tissue is expected to be the hypothalamus where foodintake and lipogenic state are regulated, a similar model is to injectthe test article directly into the brain (e.g. i.c.v. injection vialateral or third ventricles, or directly into specific hypothalamicnuclei (e.g. arcuate, paraventricular, perifornical nuclei). The sameparameters as above could be measured, or the release ofneurotransmitters that are known to regulate feeding or metabolism couldbe monitored (e.g. NPY, galanin, norepinephrine, dopamine, β-endorphinrelease).

Similar studies are accomplished in vitro using isolated hypothalamictissue in a perifusion or tissue bath system. In this situation, therelease of neurotransmitters or electrophysiological changes ismonitored.

The compounds are active in at least one of the above biological testsand are anti-obesity agents. As such, they are useful in treatingobesity and those disorders implicated by obesity. However, the proteinsare not only useful as therapeutic agents; one skilled in the artrecognizes that the proteins are useful in the production of antibodiesfor diagnostic use and, as proteins, are useful as feed additives foranimals. Furthermore, the compounds are useful for controlling weightfor cosmetic purposes in mammals. A cosmetic purpose seeks to controlthe weight of a mammal to improve bodily appearance. The mammal is notnecessarily obese. Such cosmetic use forms part of the presentinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note="Xaa at position 4 of SEQ ID                     NO: 1 is Gln or Glu;"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /note="Xaa at position 7 of SEQ ID                     NO: 1 is Gln or Glu;"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 22                                                              (D) OTHER INFORMATION: /note="Xaa at position 22 of SEQ                       ID NO: 1 is Asn, Asp or Gln;"                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 27                                                              (D) OTHER INFORMATION: /note="Xaa at position 27 of SEQ                       ID NO: 1 is Thr or Ala;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 28                                                              (D) OTHER INFORMATION: /note="Xaa at position 28 of SEQ                       ID NO: 1 is Gln, Glu or absent;"                                              (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa at position 34 of SEQ                       ID NO: 1 is Gln or Glu;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 54                                                              (D) OTHER INFORMATION: /note="Xaa at position 54 of SEQ                       ID NO: 1 is Met, methionine sulfoxide, Leu, Ile,                              Val, Ala, or Gly;"                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 56                                                              (D) OTHER INFORMATION: /note="Xaa at position 56 of SEQ                       ID NO: 1 is Gln or Glu;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 62                                                              (D) OTHER INFORMATION: /note="Xaa at position 62 of SEQ                       ID NO: 1 is Gln or Glu;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 63                                                              (D) OTHER INFORMATION: /note="Xaa at position 63 of SEQ                       ID NO: 1 is Gln or Glu;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 68                                                              (D) OTHER INFORMATION: /note="Xaa at position 68 of SEQ                       ID NO: 1 is Met, methionine sulfoxide, Leu, Ile,                              Val, Ala, or Gly;"                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 72                                                              (D) OTHER INFORMATION: /note="Xaa at position 72 of SEQ                       ID NO: 1 is Asn, Asp or Gln;"                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 75                                                              (D) OTHER INFORMATION: /note="Xaa at position 75 of SEQ                       ID NO: 1 is Gln or Glu;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 78                                                              (D) OTHER INFORMATION: /note="Xaa at position 78 of SEQ                       ID NO: 1 is Asn, Asp or Gln;"                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 82                                                              (D) OTHER INFORMATION: /note="Xaa at position 82 of SEQ                       ID NO: is Asn, Asp or Gln;"                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 100                                                             (D) OTHER INFORMATION: /note="Xaa at position 100 of SEQ                      ID NO: 1 is Gln, Trp, Tyr, Phe, Ile, Val, or Leu;"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 130                                                             (D) OTHER INFORMATION: /note="Xaa at position 130 of SEQ                      ID NO: 1 is Gln or Glu;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 134                                                             (D) OTHER INFORMATION: /note="Xaa at position 134 of SEQ                      ID NO: 1 is Gln or Glu;"                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 136                                                             (D) OTHER INFORMATION: /note="Xaa at position 136 of SEQ                      ID NO: 1 is Met, methionine sulfoxide, Leu, Ile,                              Val, Ala, or Gly;"                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 138                                                             (D) OTHER INFORMATION: /note="Xaa at position 138 of SEQ                      ID NO: 1 is Trp or Gln;"                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ValProIleXaaLysValXaaAspAspThrLysThrLeuIleLysThr                              151015                                                                        IleValThrArgIleXaaAspIleSerHisXaaXaaSerValSerSer                              202530                                                                        LysXaaLysValThrGlyLeuAspPheIleProGlyLeuHisProIle                              354045                                                                        LeuThrLeuSerLysXaaAspXaaThrLeuAlaValTyrXaaXaaIle                              505560                                                                        LeuThrSerXaaProSerArgXaaValIleXaaIleSerXaaAspLeu                              65707580                                                                      GluXaaLeuArgAspLeuLeuHisValLeuAlaPheSerLysSerCys                              859095                                                                        HisLeuProXaaAlaSerGlyLeuGluThrLeuAspSerLeuGlyGly                              100105110                                                                     ValLeuGluAlaSerGlyTyrSerThrGluValValAlaLeuSerArg                              115120125                                                                     LeuXaaGlySerLeuXaaAspXaaLeuXaa                                                130135                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note="Xaa at position 1 of SEQ ID                     NO: 2 is Gln or Glu."                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       XaaLeuAspLeuSerProGlyCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGGGCATATGAGGGTACCTATCCAGAAAGTCCAGGATGACAC42                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGGGGGATCCTATTAGCACCCGGGAGACAGGTCCAGCTGCCACAACAT48                            __________________________________________________________________________

We claim:
 1. A protein comprising the amino acids of position 95 throughposition 138 of SEQ ID NO: 1 properly crosslinked to SEQ ID NO: 2, or apharmaceutically acceptable salt thereof.
 2. A protein of claim 1,wherein the amino acids of positions 93 through 138 of SEQ ID NO: 1 areproperly crosslinked to SEQ ID NO:
 2. 3. A protein of claim 1, whereinthe amino acids of positions 72 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 4. A protein of claim 1, wherein the aminoacids of positions 62 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 5. A protein of claim 1, wherein the aminoacids of positions 54 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 6. A protein of claim 1, wherein the aminoacids of positions 42 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 7. A protein of claim 1, wherein the aminoacids of positions 36 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 8. A protein of claim 1, wherein the aminoacids of positions 34 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 9. A protein of claim 1, wherein the aminoacids of positions 21 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 10. A protein of claim 1, wherein the aminoacids of positions 16 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 11. A protein of claim 1, wherein the aminoacids of positions 12 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 12. A protein of claim 1, wherein the aminoacids of positions 6 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 13. A protein of claim 1, wherein the aminoacids of positions 3 through 138 of SEQ ID NO: 1 are properlycrosslinked to SEQ ID NO:
 2. 14. A protein of claim 1, wherein SEQ IDNO: 1 is properly crosslinked to SEQ ID NO:
 2. 15. A protein of claim14, wherein:Xaa at position 4 of SEQ ID NO: 1 is Gln; Xaa at position 7of SEQ ID NO: 1 is Gln; Xaa at position 22 of SEQ ID NO: 1 is Asn; Xaaat position 27 of SEQ ID NO: 1 is Thr; Xaa at position 28 of SEQ ID NO:1 is Gln; Xaa at position 34 of SEQ ID NO: 1 is Gln; Xaa at position 54of SEQ ID NO: 1 is Met; Xaa at position 56 of SEQ ID NO: 1 is Gln; Xaaat position 62 of SEQ ID NO: 1 is Gln; Xaa at position 63 of SEQ ID NO:1 is Gln; Xaa at position 68 of SEQ ID NO: 1 is Met; Xaa at position 72of SEQ ID NO: 1 is Asn; Xaa at position 75 of SEQ ID NO: 1 is Gln; Xaaat position 78 of SEQ ID NO: 1 is Asn; Xaa at position 82 of SEQ ID NO:1 is Asn; Xaa at position 100 of SEQ ID NO: 1 is Trp; Xaa at position130 of SEQ ID NO: 1 is Gln; Xaa at position 134 of SEQ ID NO: 1 is Gln;Xaa at position 136 of SEQ ID NO: 1 is Met; Xaa at position 138 of SEQID NO: 1 is Trp; and Xaa at position 1 of SEQ ID NO: 2 is Gln.